The general objective of this proposal is to utilize the fluorescence activated cell sorter (FACS) together with specific indirect immunofluorescent reagents to automate the speciation of bacteria from dental plaque. The FACS instrument analyzes scattered and fluorescent light from individual cells in suspension, much the same as a microscopist might, but cells are analyzed at rates up to 5,000 cells per second and a computer generates statistically analyzed histograms of light scatter parameters for cell populations. Specific aims include preparing pure cultures of bacteria frequently associated with periodontal diseases and using these bacteria as immunogens for producing rabbit antisera. Controlled studies will optimize indirect immunofluorescent staining procedures for each bacterial species for analysis by FACS and fluorescence microscopic methods. A FACS generated morphologic profile will also be generated for each bacterial species. Defined bacterial mixtures, including some formulated to simulate plaque from specific periodontal diseases, will be analyzed using FACS and fluorescent microscopic techniques to quantitate levels of specific bacteria within the mixture. The results will be statistically compared to the actual composition of the bacterial mixtures. Completion of this study will develope a method for rapid, accurate and reproducible quantitative speciation of subgingival bacteria without culturing or concern for preserving bacterial viability. The FACS method eliminates much of the subjectivity involved in interpretation of fluorescent staining of bacteria and increases the number of cells which are evaluated. A FACS quantitative bacterial speciation could be used to monitor periodontal sites during diagnosis and to evaluate progress of periodontal therapy. Bacterial species enumeration using FACS should also be useful in the diagnosis of other infectious diseases using samples from a variety of body sites. Our preliminary results indicate that the FACS system analysis of bacteria will provide information on the general morphology of bacteria and permit species specific enumeration of bacteria within mixed populations based upon specific immunofluorescent staining.